Immunoassay for carbohydrate antigenic determinant

ABSTRACT

An immunoassay for a carbohydrate antigenic determinant, such as CA 19-9, is disclosed in which the primary antigen-antibody reaction is carried out under acidic conditions.

This is a continuation of co-pending application Ser. No. 600,551, filedon Apr. 6, 1984, now abandoned.

DESCRIPTION

1. Technical Field

This invention is in the field of immunology and particularly relates toan immunoassay for detecting a carbohydrate antigenic determinant.

2. Background Art

Monoclonal antibodies are antibodies which have been produced by a cellline cloned from a single antibody producing cell. Monoclonal antibodiesare extraordinarily pure, uniform and reproducible since each antibodyis effective against a single antigenic determinant.

Monoclonal antibodies can be obtained in significant quantities fromhybridoma cells. Hybridoma cells are fused cells resulting from thefusion of antibody producing cells with tumor cells. The initial workrelating to the production of such hybridoma cells was done by CesarMilstein and George Kohler employing mouse myeloma cells with spleencells taken from mice immunized with sheep red blood cells. See, Kohleret al., Eur. J. Immunol., 6, 511-19 (1976); Kohler et al., Nature, 256,495-7 (1975); and Milstein, Scientific American, 243 (4), 66-74 (1980).

More recently, Hilary Koprowski and colleagues have extended theoriginal hybridoma work by developing hybridoma cell lines capable ofproducing monoclonal antibodies against specific viruses and tumors.See, U.S. Pat. Nos. 4,196,265 and 4,172,124, respectively. Wands andZurawski further extended hybridoma technology to produce hybridoma celllines capable of producing monoclonal antibodies to hepatitis virus. SeeU.S. Pat. No. 4,271,145. It has also been recently discovered thatmonoclonal IgM antibodies produced from hybridoma cell lines can beemployed in certain immunoassays to improve the sensitivity andspecificity thereof. See, U.S. application Ser. No. 188,735, filed Sept.19, 1980, in the names of Jack R. Wands, Vincent R. Zurawski, and HubertJ. P. Schoemaker.

Even more recently, a monoclonal antibody, designated 1116 NS 19-9(hereinafter "19-9 antibody"), was developed by Koprowski et al. byimmunization of BALB/c mice with a human colorectal cancer cell line,SW1116. See Koprowski, H., Steplewski, Z., Mitchell, K., Herlyn, M.,Herlyn, D., and Fuhrer, P., Colorectal carcinoma antigens detected byhybridoma antibodies, Somatic Cell Genetics, 5, 957-972, (1979). It hasbeen shown that the 19-9 antibody reacts with a carbohydrate antigenicdeterminant (hereinafter referred to as "CA 19-9") which has beenidentified as a sialylated lacto-N-fucopentaose II, an oligosaccharidewhich shares structural features with Lewis blood group substances. SeeMagnani, J., Nilsson, B., Brockhaus, M., Zopf, D., Steplewski, Z.,Koprowski, H., Ginsburg, V., The antigen of a tumor-specific monoclonalantibody is a ganglioside containing sialylated lacto-N-Fucopentaose II,Fed. Proc. 41, 898, (1982).

In early studies using a competition radioimmunoassay, the 19-9 antibodywas shown to have high sensitivity in identifying patients withgastrointestinal adenocarcinosmas and to have high specificity fornormal individuals. See Koprowski, H., Herlyn, H., Steplewski, Z., andSears, H. F., Specific antigen in serum of patients with coloncarcinoma, Science, 212, 53, (1981); and, Herlyn, M., Clark, W. H.,Mastrangelo, J. J., Guerry, D., Elder, D. E., Larossa, D., Hamilton, R.,Bondi, E., Tutkill, R., Steplewski, Z., Koprowski, H., Specific antigensto colorectal carcinoma in sera of patients are detected by monoclonalantibodies, Cancer Res. 40, 3602-3609, (1982). The CA 19-9 epitope hasalso been identified on a glycolipid extracted from SW1116 cells andfrom meconium, as well as on a glycoprotein from SW1116 cells. SeeMagnani, J., Brockhaus, M., Smith, D., Ginsburg, V., Blaszcyk, M.,Mitchell, D., Steplewski, Z., and Koprowski, H.; A Monoclonal AntibodyDefined Antigen of colon Carcinoma, Science 212, 55, (1981).

DISCLOSURE OF THE INVENTION

This invention relates, in its broadest aspects, to immunoassays fordetecting carbohydrate antigenic determinants.

In one embodiment, a forward sandwich immunoassay is employed to detectthe CA 19-9 antigen. In this assay, a patient sample containing theantigen is initially incubated with a solid-phase immunoadsorbentcontaining immobilized 19-9 antibody. Incubation is continued for asufficient period of time to allow antigen in the patient sample to bindto immobilized antibody on the solid-phase immunoadsorbent. After thisfirst incubation, the solid-phase immunoadsorbent is separated from theincubation mixture and washed to remove excess antigen and otherinterfering substances, such as non-specific binding proteins, whichalso may be present in the patient sample. The solid-phaseimmunoadsorbent containing antigen bound to immobilized antibody issubsequently incubated for a second time with labeled antibody capableof binding to CA 19-9 antigen. After the second incubation, another washis performed to remove unbound labeled antibody from the solid-phaseimmunoadsorbent thereby removing non-specifically bound labeledantibody. Labeled antibody bound to the solid-phase immunoadsorbent isthen detected and the amount of labeled antibody detected serves as adirect measure of the amount of CA 19-9 antibody present in the originalpatient sample.

The immunoassay is preferably carried out under conditions whereby theantigen-antibody reaction occurs at acidic pH. This is because it hasbeen surprisingly found that higher signal/noise ratios can be achievedunder such conditions.

The resulting assay has high sensitivity for patients withgastrointestinal adenocarcinomas, especially pancreatic cancer, and hasvery high specificity, even among patients with benign gastrointestialdisease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a standard curve showing counts per minute of radiation fromthe labeled antibody as a function of known amounts of CA 19-9 antigenpresent.

FIG. 2 is a depiction of the CA 19-9 concentration in patient sera forpatients having different types of cancer or benign diseases.

FIG. 3 is a plot illustrating the frequency of various CA 19-9concentrations in sera from normal blood bank donors.

BEST MODE FOR CARRYING OUT THE INVENTION

Although most of the experimental work described below was carried outat a pH of 4.5, it should be recognized that other acidic pHs could havebeen employed. This would especially be true for carbohydrate antigenicdeterminants other than CA 19-9, or antibodies other than the 19-9antibody. With regard to detecting CA 19-9 antigen with 19-9 antibody,the preferred pH range was found to be from about 2.5 to about 6.5, andit is especially preferred to operate at a pH of about 4.5.

Similarly, the experimental work described herein relates to a forwardsandwich assay. Nevertheless, it is believed that carbohydrate antigenicdeterminants can be determined in many cases employing acidic conditionswith reverse sandwich assays or simultaneous sandwich assays.

In addition to labeling antibody with radio-active labels, other labels,such as fluorescent materials, could also be employed. Further, enzymelabels could be employed, in which case detection could be achieved by acolorimetric method employing a competing substrate for the enzymelabel.

The forward sandwich assay for CA 19-9 will now be more specificallyillustrated by way of the following example.

EXAMPLE

Murine hybridoma 1116-NS-19-9 was obtained from Dr. Z. Steplewski(Wistar Institute, Philadelphia, PA). Hybridoma cells were grown intissue culture and were used to prepare ascitic fluid in pristane primedBALB/c mice. IgG was purified from ascites by affinity chromatography onprotein A-Sepharose (Pharmacia) using elution at pH 4.0. Purity of theisolated IgG was demonstrated by SDS polyacrylamide gel electrophoresis,isoelectric focusing and immunoelectrophoresis.

Purified antibody was radioiodinated using ¹²⁵ I labeledN-hydroxysuccinimide ester of p-hydroxyliodophenylpropionic acid,Bolton-Hunter reagent, using the manufacturer's recommended procedure.Specific activity of the ¹²⁵ I IgG ranged from 5 to 15 μCi/μgm.Polystyrene beads (1/4"; Precision Plastic Ball Co., Chicago, Ill.) werecoated with purified antibody and dried.

Experimental samples were assayed in duplicate using a "forwardsandwich" radioimmunometric assay. One hundred microliters of sera,standard solutions or controls were mixed with 100 μl of buffer (5 mg/mlbovine serum albumin, Armour Pharmaceutical, So. Plainfield, NJ; 50 mMsodium citrate; 1 mM ethylene diamine tetracetic acid; pH 3.0) in areaction tray. An antibody coated bead was added and samples wereincubated at 37° for 3 hours. Beads were washed with deionized water,then 200 μl of ¹²⁵ I labeled 19-9 antibody (130,000 dpm; 5 mg/ml bovineserum albumin; 50 mM sodium citrate; pH 4.5) was added. Samples werethen incubated for 3 hours at room temperature (18°-22°). Beads werewashed and then counted in a gamma counter.

Experimental results were converted into units by comparison with astandard curve prepared as follows. Partially purified CA 19-9 wasdiluted into pooled reconstituted human serum to give concentrationsbetween 6 and 160 units/ml. One hundred microliters of standards weremixed with 100 μl of buffer and an 19-9 antibody coated bead for 3 hoursat 37°. Beads were washed with dH₂ O then ¹²⁵ I labeled 19-9 antibody(130,000 dpm) was added and incubated for 3 hours at 20° C. Afterwashing, ¹²⁵ I bound to beads was determined using a gamma counter. Theresulting standard curve is illustrated as FIG. 1.

Frozen coded samples from the National Cancer Institute (NCI) serum bankwere provided by Dr. R. Herberman (NCI). Serum was carefully preparedand frozen continuously at -70°. Clinical data were compiled fromreports provided by Dr. V. Go (Mayo Clinic). Additional serum samplesfrom random healthy blood bank donors were obtained from Dr. J.Menitove, Southeastern Blood Center of Milwaukee, Wis. All donorsqualified as blood donors, and were negative when tested for hepatitis Bsurface antigen. Data concerning age and sex were provided by Dr.Menitove.

The levels of CA 19-9 were determined by plotting [¹²⁵ I]-19-9 boundversus the concentration of CA 19-9 in standard solutions. The CA 19-9concentration of each standard was specified in arbitrary units. Asshown in FIG. 1, the amount of ¹²⁵ I labeled 19-9 antibody boundapproximated a linear function of concentration up to at least 80units/ml. The intra- and interassay coefficients of variation were 5-12%in the range between 5 and 120 units/ml and the minimum detectable dosewas 1.5 units/ml.

The concentration of CA 19-9 in sera from a coded panel from the MayoClinic-NCI and in sera from blood bank donors was determined using theCA 19-9 assay. Four hundred and eleven samples from the Mayo Clinic-NCISerum Bank were assayed. As shown in FIG. 2, the level of CA 19-9 in theserum of cancer patients was frequently higher than that in the serum ofnormal individuals or of patients with benign diseases. Using anarbitrary value of 37 units/ml to discriminate between positive andnegative samples, sera from 19 of 25 (74%) pancreatic cancer patientswere found to contain elevated levels of CA 19-9. Among 75 patients withcolorectal cancer, 12 (16%) had CA 19-9 levels above 37 units/ml,including 6 of 23 (26%) having distant metastasis, 4 of 25 (16%) Dukes Band C, and 2 of 27 (7%) Dukes A Disease. Elevated levels of CA 19-9 werealso observed in 10 of 31 (32%) sera of patients with othergastrointestinal malignancies (Table 1). High concentrations wereusually associated with adenocarcinomas of the hepatobiliary system(gall bladder, ampulla of vater, bile ducts); a gastric adenocarcinomaand a hepatoma were also above 37 units/ml. Additionally, 4 of 25 (16%)sera from lung cancer patients, 1 of 25 (4%) sera from breast cancerpatients and 3 of 24 (12%) sera from other cancer patients (Table I) hadelevated CA 19-9 levels. In two of these cases (357; 94 units/ml) thesite of the primary tumor was unknown and in the third (39 units/ml) thesite of the primary was the thyroid. Among 131 patients with benigndiseases, only one (0.8%), a 47 year old male with interstitialpneumonitis of unknown etiology had elevated CA 19-9 (50.7 units/ml). Asshown in Table 2, the benign disease group represented a broad range ofgastrointestinal and non-gastrointestinal diseases, including 11patients with benign pancreatic diseases, 52 patients with othergastrointestinal diseases, and 68 with non-gastrointestinal diseases.Only 1 of 75 (1.3%) normal persons had elevated CA 19-9 (40.7 units/ml).

The mean CA 19-9 levels obtained for patient sera from all groups in thepanel are presented in Table 3. The highest CA 19-9 levels were detectedin sera of patients with pancreatic carcinoma (266±266 units/ml). CA19-9 levels averaged 8.4±7.6 units/ml in normal individuals, 6.5±6.2units/ml in patients with benign gastrointestinal disorders, 7.1±5.4units/ml with benign pancreatic disease, and 8.6±9.5 units/ml withbenign non-enteric disease. No statistically significant differences inthese values could be found when patients were grouped according to ageor fasting or nonfeeding, i.e. the CA 19-9 levels appeared to beindependent of these parameters in the patient groups studied (data notshown).

Sera obtained from randomly selected, healthy blood bank donors wereevaluated using the CA 19-9 assay. As shown in FIG. 3, the mean for thenormals was 8.7±7.4 units/ml (range 0-107 units/ml). Only 0.5% of thesesamples contained CA 19-9 at levels in excess of 37 units/ml. Inaddition, there was no statistically significant relationship betweenthe age of the donors and the level of CA 19-9 in their sera. Small butstatistically significant differences were observed between males andfemales. Females between 20 and 49 years of age had slightly higherlevels than males. No further information could be obtained concerningthe donors whose sera had elevated CA 19-9 levels.

The sensitivity of the CA 19-9 RIA for pancreatic cancer (74%) and thespecificity among normal individuals (99.5%), and among patients with awide variety of benign diseases (99.2%) were excellent. These benigndiseases included benign pancreatic diseases, diabetes, arthritis andinflammatory gastrointestinal disorders.

CA 19-9 levels above 37 units/ml were observed in several patients withother gastrointestinal malignancies, particularly of the hepatobiliarysystem. Because the CA 19-9 antigen is present in normal tissues,especially the epithelial cells lining the ductules in the liver andpancreas, more extensive studies among patients with hepatobiliary tractdisorders and pancreatitis will be required to assess reactivity inthese patients.

CA 19-9 levels above 37 units/ml were observed in relatively fewpatients with colorectal cancer. Only 7% of patients with Dukes A, 16%of patients with Dukes B or C and 26% of patients with advancedmetastatic colorectal disease had elevated CA 19-9 values.

Sera from some patients with non-gastrointestinal malignancies,especially lung tumors, had elevated levels of CA 19-9. In the fourpositive lung cancer patients, there was no apparent relationship totumor type, grade or stage: Three were adenocarcinomas, grades 3 and 4,stages I, II, and III. The fourth was a squamous cell carcinoma, grade3, stage I. Only one breast adenocarcinoma and one thyroid papillarycarcinoma had elevated CA 19-9 levels.

To determine the effect of pH, a series of forward sandwich assays, asdescribed above, were performed on samples prepared from one serumsample (PC 2). CA 19-9 antigen was purified from human ascites and thendiluted into normal human serum. Samples were prepared by mixingserum-diluted CA 19-9 with either citrate or phosphate buffer andsufficient hydrochloric acid to produce the desired pH. The results arepresented in Table 4.

                  TABLE 1                                                         ______________________________________                                        CA 19-9 Levels in Sera From Patients With                                     Miscellaneous Cancer                                                                               CA 19-9                                                  Diagnosis            (units/ml)                                               ______________________________________                                        Ampulla of Vater     395.7                                                                         41.6                                                                          4.2                                                      Stomach              535.6                                                                         7.9                                                                           0.0                                                      Hepatoma             333.5                                                                         10.0                                                     Hepatic and Cystic Duct Cancer                                                                     63.2                                                     Cancer Bile Duct     151.5                                                                         112.8                                                                         23.3                                                     Gall Bladder         640.8                                                                         528.2                                                                         5.0                                                      Appendix, Carcinoid  0.0                                                      Ileum                9.6                                                                           6.1                                                                           2.7                                                      Duodenum Cancer      27.3.                                                                         8.2                                                                           0.1                                                      Esophagus            45.1                                                                          7.7                                                                           6.9                                                                           6.9                                                                           6.2                                                                           5.4                                                                           4.3                                                                           3.3                                                      Barrett's Esophagus  5.4                                                      Primary unknown      357.6                                                                         93.6                                                                          10.3                                                                          0.0                                                      Burkitt's Lymphoma   17.9                                                     Lymphoma (Remission) 20.8                                                     Leukemia             8.6                                                                           7.1                                                      Hodgkin's Disease    8.2                                                      Malignant Histiocytoma                                                                             3.2                                                      Plasmacytoma (pulmonary)                                                                           0.0                                                      Kidney               16.5                                                                          5.9                                                      Bladder              11.2                                                     Testicular           9.0                                                      Prostate             3.7                                                      Thyroid              39.0                                                                          0.0                                                      Malignant Melanoma   8.4                                                      Cancer Nasopharynx   0.0                                                      Synovial Cell Sarcoma                                                                              2.6                                                      Cancer Retroperitoneum                                                                             3.3                                                      Osteogenic Sarcoma   2.5                                                      Liposarcoma (thigh)  1.2                                                      ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        CA 19-9 Levels In Sera From Patients With Benign Diseases                                         CA 19-9                                                   Diagnosis           (units/ml)                                                ______________________________________                                        Arthritis, Degenerative                                                                           21.2                                                                          9.5                                                                           5.0                                                                           0.0                                                       Arthritis, Osteo    15.1                                                                          5.3                                                       Arthritis, Rheumatoid                                                                             5.7                                                                           3.9                                                                           0.2                                                       Arthritis           5.4                                                                           4.7                                                                           4.4                                                       Degenerative Joint Disease                                                                        16.0                                                                          9.1                                                                           4.4                                                                           1.9                                                       Hypothyroidism      4.2                                                       Thyroiditis         15.9                                                      Thyroiditis, Hashimoto's                                                                          15.1                                                                          5.4                                                       Graves' Disease     14.9                                                                          5.6                                                       Fibrocystic Disease (breast)                                                                      13.7                                                                          5.0                                                       Gynecomastia        5.4                                                                           4.2                                                       Psoriasis           9.4                                                       Pancreatitis                                                                  Fatty Liver         28.3                                                      Cirrhosis           25.1                                                                          13.7                                                                          7.2                                                                           6.7                                                                           4.5                                                                           2.4                                                       Chronic Ulcerative Colitis                                                                        15.7                                                                          3.8                                                                           3.6                                                                           1.5                                                                           0.0                                                                           0.0                                                       Diarrhea, unknown etiology                                                                        14.6                                                                          13.2                                                      Irritable Colon/Bowel                                                                             6.9                                                                           6.3                                                                           4.8                                                                           4.1                                                       Crohn's Disease     6.8                                                                           0.0                                                       Achalasia           5.0                                                                           0.2                                                       Post Vagotomy Diarrhea                                                                            3.0                                                       Pyloric stenosis    2.6                                                       Pneumonitis (interstitial,                                                                        50.7                                                      etiology unknown)                                                             Systemic Mast Cell Disease                                                                        35.3                                                      (C.O.P.D.)                                                                    Pemphigus - vulgaris                                                                              32.1                                                      Low Blood Pressure  29.2                                                      Diabetes            22.5                                                                          22.4                                                                          13.4                                                                          5.0                                                                           4.3                                                                           0.0                                                       Pericardial Cyst    19.1                                                      Cholelithiasis      14.5                                                      Hepatitis, Chronic, Active                                                                        13.4                                                                          10.2                                                                          8.3                                                                           0.0                                                       Malabsorption Syndrome                                                                            13.2                                                      Polyp               12.3                                                      Ulcer(s)            11.3                                                                          8.3                                                                           3.2                                                                           3.0                                                                           2.7                                                                           2.1                                                                           1.7                                                                           0.0                                                                           0.0                                                       Gastritis, Duodenitis                                                                             10.3                                                                          8.9                                                                           6.3                                                                           5.0                                                                           2.0                                                       Intermediate Gastric                                                                              7.5                                                       Motility Disorder   3.9                                                       Gastric Hypertension,                                                                             7.3                                                       Abdominal Pain                                                                Granuloma           11.0                                                                          10.2                                                                          4.4                                                                           4.3                                                                           2.9                                                       Benign Bone Cyst    8.3                                                       Pregnancy           6.8                                                       Thrombocytopenic purpura                                                                          2.3                                                       Chronic alcoholism  1.3                                                       Fibrosis            4.4                                                       Turner's Variant    0.0                                                       Dyspepsia           0.0                                                       Unknown Abdominal Pain                                                                            0.0                                                       Alcohol Abuse       4.4                                                                           4.1                                                       Menometrorrhagia    3.6                                                       Sarciodosis         3.1                                                                           3.0                                                                           0.0                                                       Hyperplastic Mucosa 3.5                                                       Polyps, cervical    2.0                                                       Scoliosis           2.7                                                       Hematoma            4.9                                                       Thornwaldt's Disease                                                                              4.1                                                       Aneurysm            3.1                                                       Dumping Syndrome    0.0                                                       Benign Prostatic    4.1                                                       Hypertrophy                                                                   Friction Blisters   2.7                                                       Mixed Connective Tissue                                                                           1.8                                                       Disease                                                                       ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        AVERAGE CA 19-9 CONCENTRATION                                                 IN PATIENT SERA                                                               Group      n       Mean ± S.D.                                                                           Percent Positive.sup.a                          ______________________________________                                        A. Cancers                                                                    Pancreas   25      266 ± 266                                                                             74                                              Colon                                                                         Metastatic 23      81.9 ± 168                                                                            26                                              Dukes B & C                                                                              25      59.8 ± 123                                                                            16                                              Localized  27      10.5 ± 14.2                                                                           7                                               GI (misc.) 31      97.3 ± 182                                                                            32                                              Lung       25      52.8 ± 109                                                                            16                                              Breast     25      10.3 ± 12                                                                             4                                               Other      24      26.7 ± 73.1                                                                           12                                              B. Benign diseases                                                            Pancreatic 11      7.1 ± 5.4                                                                             0                                               Other GI   52      6.5 ± 6.2                                                                             0                                               Non-GI     68      8.6 ± 9.5                                                                             1.4                                             C. Normals                                                                    Normals    75      8.4 ± 7.6                                                                             1.3                                             ______________________________________                                         .sup.a Positive 37 units/ml                                              

                  TABLE 4                                                         ______________________________________                                        Effect of Buffer pH on CA 19-9 Detected                                       Buffer pH     CPM.sup.125 I-19-9                                              ______________________________________                                        1.5           4680                                                            2.0           5970                                                            2.5           8880                                                            3.0           9480                                                            3.5           10860                                                           4.0           23500                                                           4.5           31100                                                           5.0           26400                                                           5.5           22800                                                           6.0           14800                                                           6.5           8260                                                            7.0           4580                                                            7.5           2870                                                            8.0           2180                                                            ______________________________________                                    

Industrial Applicability

The invention described herein is useful in the detection ofcarbohydrate antigenic determinants, such as CA 19-9. The assay can beemployed by hospitals or clinical laboratories for the early detectionof tumors, particularly pancreatic cancer tumors.

Equivalents

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, many equivalents to the specificembodiments to the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. In a solid phase, forward immunometric assay for thecarbohydrate antigenic determinant CA19-9, comprising reacting themonoclonal 1116-NS-19-9 with the CA-19-9 antigen, the improvementwherein the reaction between the CA 19-9 antigen and the antibody1116-NS-19-9 is carried out at an acidic pH.
 2. An improvement of claim1, wherein the acidic pH is between a pH of 2.5 and
 6. 3. An improvementof claim 1, wherein the acidic pH is a pH of about 4.5.
 4. In a forwardimmunometric assay for the carbonhydrate antigenic determinant CA 19-9,comprising the steps of incubating a liquid sample with a solid phaseimmunoadsorbent containing monoclonal antibody 1116-NS-19-90 for aperiod sufficient to allow CA 19-9 antigen in the sample to bind to theantibody; separating the immunoadsorbent from the sample; incubating theimmunoadsorbent with a labeled antibody 1116-19-9 which binds CA 19-9antigen; separating the immunoadsorbent from the labeled antibody andquantitating label associated with the immunoadsorbent to obtain measureof the amount of CA 19-9 in the sample;the improvement wherein theincubation steps are carried out at an acidic pH.
 5. An improvement ofclaim 4, wherein the acidic pH is between a pH of 2.5 and
 6. 6. Animprovement of claim 4, wherein the acidic pH is a pH of about 4.5.